Microbial sensor system for the assessment of subsurface environments

ABSTRACT

A microbial sensor, system, and method that can be used to determine a chemical environment and/or substrate concentrations in anaerobic or aerobic environments, such as soils, sediments and ground waters, are disclosed. An exemplary system uses one or more (e.g., inert) measurement electrodes and a reference electrode. The reference electrode can include an electrode exposed to atmospheric oxygen (e.g., a cathode) or an electrode exposed to stable anaerobic or aerobic conditions. The exemplary microbial sensor system measures open-circuit voltage to characterize the chemical (oxidizing or reducing) environment and/or recovery voltage to measure substrate concentrations in the subsurface.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.15/237,230, filed Aug. 15, 2016, entitled “Microbial Sensor System forthe Assessment of Subsurface Environments,” which claims the benefit ofU.S. Provisional Application No. 62/205,254, filed Aug. 14, 2015,entitled “Microbial Sensor System for the Assessment and Remediation ofEnvironmental Contamination in Anaerobic Environments,” U.S. ProvisionalApplication No. 62/263,362, filed Dec. 4, 2015, entitled“Field-Deployable Microbial Fuel Cell Sensor System for theCharacterization of Environmental Contamination in Aerobic and AnaerobicEnvironments,” and U.S. Provisional Application No. 62/308,680, filedMar. 15, 2016, entitled “Field-Deployable Microbial Fuel Cell SensorSystem for the Characterization of Environmental Contamination inAerobic and Anaerobic Environments,” the contents of which areincorporated herein by reference to the extent such contents do notconflict with the present disclosure.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support by the Office of Sciencegrant DE-FOA-0001405 awarded by the Department of Energy. The governmenthas certain rights in the invention.

FIELD OF INVENTION

The present disclosure generally relates to microbial sensors, systems,and to methods of using the sensors and systems. More particularly, thepresent disclosure relates to sensors and systems that employopen-circuit voltage and/or recovery voltage measurements to provideinformation regarding microbial activity.

BACKGROUND OF THE DISCLOSURE

Microbial fuel cells were developed primarily for the conversion ofwaste products (sewage, farming wastes, etc.) into electrical energy.However, other applications of microbial fuels cells include use asanalytical sensors and bioremediation. The primary difference in themicrobial fuel cells for energy production and/or bioremediationapplications versus the analytical applications is the magnitude of thecurrent generated. Energy production and bioremediation applicationgenerally require anodes and cathodes with large surfaces to increasethe production of current, whereas high current is not of primaryimportance for analytical sensor applications.

Microbial fuel cell technology can be roughly divided into two basictypes of designs: 1) reactor designs, and 2) probe designs. The twodesigns have been used for energy production, bioremediation, andanalytical applications. The differences between the two designs arebased on: 1) the placement and orientation of the anode and cathode, 2)method of substrate (oxidizable organic materials) introduction to theanode, and 3) the method of providing the ultimate electron acceptor(e.g., oxygen, ferricyanide, and the like) to the cathode.

Reactor Designs

A reactor design typically incorporates the anode inside of an anodechamber where the wastewater or natural waters are flowed through thechamber. The cathode design is considerably more varied and includes: 1)air cathodes, 2) cathodes located in a separate cathode chamber, and 3)poised electrodes. Air cathodes are incorporated into the wall of theanode chamber and allow the diffusion of atmospheric oxygen through asemi-permeable membrane to a cathode located within the anode chamber.Cathodes located in a separate cathode chamber are separated from theanode chamber using a semi-permeable membrane. The cathode chamber istypically filled with an oxygenated aqueous solution, or alternativelyfilled with a solution (e.g., ferricyanide) capable of acceptingelectrons from the anode. The chamber design is used for energyproduction, bioremediation, and analytical applications.

The reactor design applications for analytical sensor have beenprimarily limited to applications where high concentrations (e.g.,millimolar (mM)) of organic substrates exist in wastewaters and/orsludges associated with sewage plants and solid waste disposalfacilities. One significant application for microbial sensors is thedetermination of biological oxygen demand (BOD) in wastewaters.Microbial sensors designed for the determination BOD of wastewaterstypically use a reactor design. Wastewater is transported in pipes attreatment facilities, and therefore it is convenient to divert the flowof the wastewaters through reactors for the measurement of BOD.

Typical BOD sensors measure the electrical current between the anode andcathode (or poised electrode) as the metric for BOD. Disadvantages ofsuch sensors using a reactor design for analytical applications forcharacterizing submerged sediments and natural waters include:

-   -   The reactor design requires the substrate in water to be passed        through an anode chamber. This is not a viable option if the        anode is being directly inserted in sediments, soils and        groundwater.    -   Reconfiguration of reactor designs to match the actual site        conditions is difficult and not suitable for a majority of the        sites.    -   Most reactor designs are optimized (anode and cathode size,        microbial composition, and performance) for energy production        that is not an important parameter for an analytical sensor.    -   Reactor design is not convenient for the deployment of multiple        sensors to characterize the chemical (oxidizing and reducing)        environment of a site.

The sensors rely on current measurement to determine microbialactivity/substrate concentration; such current measurement may not besensitive enough to measure desired microbial activity.

Probe Designs

Probe design generally include separate anode and cathode componentsthat are not placed into chambers. The probes are usually placed intonatural environments, or artificial ponds/digesters at wastewatertreatment facilities. The anode is placed in either anaerobic sedimentin natural environments, or at an anaerobic zone in wastewater treatmentponds/digesters. The cathode is typically placed into an oxygenated zoneabove the anaerobic zone where the anode is deployed. The probe designis used for energy production, bioremediation, or analyticalapplications.

Anode and cathode probes are used in the production of electrical powerin marine environments. In these cases, the anodic probe is buried inthe anaerobic marine sediments and the cathodic probe is positionedabove the anaerobic sediments in the oxygenated water. The typicalapplication of these benthic probes is the production of energy fornavigation buoys and other marine instrumentation. Benthic probes areprimary used for power production and not as analytical sensors.

A probe that uses three electrodes for energy production and organiccontaminant removal at wastewater or sewage treatment facilities basedon changing conditions of the organic contaminants present in the wateris disclosed in U.S. Pat. No. 9,299,999, issued in the name of Chang etal. (“the '999 Patent”). The three-electrode system was developed forenergy production and contaminant removal, not as an analytical sensor.A primary concern of the '999 Patent is the optimization of electricalcurrent in changing environments. The three-electrode system has afloating cathode and an anode placed into the sediment or sludge at thebottom of a digester. The third electrode is located in the water columnbetween the anode and cathode to serve as either and anode or cathodedepending on the water conditions.

A BOD analytical system combines the anode and the electron acceptorinto the same probe is disclosed in U.S. Pat. No. 6,113,762, issued inthe name of Kruber et al. The probe design does not use oxygen as theultimate electron acceptor, but rather uses a three-electrode system:counter electrode, microbial electrode and reference electrode with apotentiostat.

Microbial fuel sensors have been deployed in the environment to measuremicrobial activity in groundwater for bioremediation applications. Oneapplication deployed an anodic probe within a monitoring well todetermine the reduction of uranium (VI) to uranium (IV) at a sitelocated in Rifle, Colo. The cathode was located at the surface of thesite. Reagents were injected into the contaminated groundwater to inducethe reduction of uranium. The injection of reagents resulted inrelatively high substrate concentrations (e.g., on the order of mM) inthe aquifer. The electrical current was measured between the anode andcathode as the metric for acetate concentrations. The cathode was placedinto an oxidizing environment at ground surface.

A microbial sensor system was used to evaluate the operatingcharacteristics of the system when exposed to very low concentrations(e.g., on the order of micromolar (uM) or nanomolar (nM)) of substrates.The results of the investigation indicated that microbial sensors haveenvironmental applications at low substrate concentrations and/or in theevaluation of turnover rates. The electrical current was measuredbetween the anode and cathode of the system as the metric for substrateconcentration. The investigation performed in 2014 and was cited by theauthors as being the first investigation of microbial sensors beingexposed to very low concentrations of substrates. The anode was placedinto the anaerobic zone of the chamber (bottom) while the top zone ofthe chamber was oxygenated. The cathode was placed into the oxygenatedzone. The system was developed to determine if microbial sensors coulddetect current at very low concentrations in a variety of sediments, notas a practical analytical system that could be deployed in the field.

Below are illustrative disadvantages of the prior art probe designs andmethods for analytical applications of characterizing submergedsediments and natural waters.

-   -   The probe designs place the anode in the anaerobic zone        (sediment or water) but most designs place the cathode in an        aerobic zone located above the anaerobic zone within the same        pond or test chamber, or at the surface. This limits the ability        to deploy the system at sites with completely anaerobic        conditions.    -   The probes designs do allow for reconfiguration of the probes        for deployment in a variety of environments including soils,        sediments and groundwater.    -   The systems use measurement of current as the metric of        substrate concentration or turn-over rate.    -   Multiple sensor deployments require using multiple reference        electrodes.    -   The systems require use of a cathode or a poised electrode.

SUMMARY OF THE DISCLOSURE

Various embodiments of the present disclosure relate to methods andsystems for characterizing an environment, such as environments thoughtto be contaminated. While the ways in which various embodiments of thedisclosure address the drawbacks of the prior art are discussed in moredetail below, in general, the disclosure provides sensors that arerelatively sensitive (can detect very low substrate concentrations),have a relatively simple design, have a reconfigurable design—allowingfor use of the sensor in a wide variety of applications, and/or can havea relatively long lifetime in the environment, systems including thesensors, and methods of using the sensors and systems.

In accordance with various embodiments of the disclosure, a microbialsensor system includes a cathode assembly comprising a cathode in afirst environment, a conduit coupled to the cathode assembly, theconduit configured to expose the cathode to a second environment, one ormore anodes in the first environment, and a device (e.g., a device thatis part of a measurement and/or control/communication module) capable ofcharacterizing the first environment by measuring one or more of an opencircuit voltage between the anode and the cathode and a recovery voltagebetween the cathode and anode, the device coupled to and interposedbetween the cathode and the one or more anodes. In accordance withvarious aspects of these embodiments, the open circuit voltage is usedto characterize or determine a chemical environment near the anode. Forexample, a measurement or measurements of the open circuit voltage canbe used to determine whether the first environment is aerobic oranaerobic, and substrate concentrations in the first environment. Inaccordance with additional aspects, the recovery voltage can be used tocharacterize or determine a substrate concentration in the firstenvironment. In accordance with further aspects, the microbial sensorsystem further includes a float coupled to the cathode.

In accordance with further exemplary embodiments of the disclosure, amicrobial sensor system includes one or more measurement electrodes inan environment, a reference electrode in the environment, and a devicecapable of characterizing the environment by measuring one or more of anopen circuit voltage between the reference electrode and each of the oneor more measurement electrodes, and a recovery voltage the between thereference electrode and each of the one or more measurement electrodes,the device coupled to and interposed between the reference electrode andthe one or more measurement electrodes. In accordance with some aspectsof these embodiments, the reference electrode can include an inertelectrode or the most stable electrode.

In accordance with further exemplary embodiments of the disclosure, amicrobial monitoring system includes a cathode assembly comprising acathode and a permeable membrane, a conduit coupled to the cathodeassembly, the conduit configured to provide access to a secondenvironment when the cathode assembly is submerged in a firstenvironment, an anode, and a device interposed between the anode and thecathode, the device capable of measuring one or more of an open circuitvoltage and a recovery voltage between the anode and the cathode tocharacterize the first environment.

Exemplary systems described herein can additionally include calibrationmodule(s), temperature sensors, and/or various connectors.

In accordance with additional embodiments of the disclosure, a method ofmeasuring microbial activity in an environment or otherwisecharacterizing the environment includes providing a reference electrodein the environment, providing one or more measurement electrodes in theenvironment, and measuring one or more of an open circuit voltagebetween the reference electrode and the one or more measurementelectrodes and a recovery voltage between the reference electrode andthe one or more measurement electrodes to characterize the environment.In accordance with some aspects of these embodiments, the referenceelectrode can include an inert electrode or the most stable electrode.Exemplary methods additionally include measuring a current between areference electrode and a measurement electrode.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

A more complete understanding of exemplary embodiments of the presentdisclosure may be derived by referring to the detailed description andclaims when considered in connection with the following illustrativefigures.

FIG. 1 illustrates a system including anode and cathode assemblies inaccordance with at least one embodiment of the disclosure.

FIG. 2 illustrates another system including anode and cathode assembliesin accordance with at least one embodiment of the disclosure.

FIG. 3 illustrates an exemplary system deployed within a monitoring wellin accordance with yet additional embodiments of the disclosure.

FIG. 4 illustrates a system with one reference cathode and multipleanodes in accordance with at least one embodiment of the disclosure.

FIG. 5 illustrates yet another system, which includes one alternativereference electrode and multiple measurement electrodes, in accordancewith at least one embodiment of the disclosure.

FIG. 6 is a graph illustrating open circuit voltage measurements overtime using an exemplary system of the disclosure.

FIG. 7 is a graph illustrating recovery voltage over time as measuredusing a system in accordance with exemplary embodiments of thedisclosure.

FIG. 8 is a log-log graph further illustrating the recovery voltage overtime data illustrated in FIG. 7.

It will be appreciated that elements in the figures are illustrated forsimplicity and clarity and have not necessarily been drawn to scale. Forexample, the dimensions of some of the elements in the figures may beexaggerated relative to other elements to help to improve understandingof illustrated embodiments of the present disclosure.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE DISCLOSURE

The description of exemplary embodiments of methods, systems, and probesprovided below is merely exemplary and is intended for purposes ofillustration only; the following description is not intended to limitthe scope of the disclosure or the claims. Moreover, recitation ofmultiple embodiments having stated features is not intended to excludeother embodiments having additional features or other embodimentsincorporating different combinations of the stated features.

As noted above, prior microbial sensor technologies (energy production,bioremediation, analytical sensors) are primarily based on themeasurement of electrical current between an anode and a cathode. Themeasurement of constant current allows for the determination ofsubstrate concentration in a solution. In contrast, the inventorssurprisingly found that measurements of open-circuit voltage (OCV) andrecovery voltage (RV) are capable of providing information distinct fromthe measurement of constant current and can use less sensitiveinstrumentation to provide meaningful information regarding substratesand concentrations thereof that are or may be present in an environment.

Furthermore, the prior-art techniques generally do not address designsand methods for deployment of microbial sensors in the environmentincluding:

-   -   Applications where the cathode is deployed below the static        water level in anaerobic environments (groundwater and submerged        soils).    -   One cathode (serving as a reference electrode) to evaluate        open-circuit voltages of multiple anodes deployed in the        environment.    -   The use of an alternative reference electrode (as a replacement        of the cathode requiring atmospheric oxygen) to evaluate the        open-circuit voltages of multiple anodes deployed in the        environment. The reference electrode can have the same design as        the anodes, but is located in a stable anaerobic environment.    -   Use of open-circuit voltage as a metric for determination of the        chemical (oxidizing/reducing conditions) environment of        submerged soils/sediments and ground waters.    -   Use of recovery voltage as a metric for the determination of        substrate concentrations and/or turn-over rates in submerged        soils/sediments and ground waters.        Various embodiments of the present disclosure include such        designs and, to the extend not inconsistent with this        disclosure, combinations of such designs.

The present disclosure relates generally to fuel cell technology beingused in analytical sensors to determine the chemical (oxidizing orreducing) characteristics and substrate concentrations in environments,such as saturated sediments and ground waters. Exemplary sensor systemscan be incorporated into a probe to allow for deployment at a variety ofenvironmental sites. The design of the exemplary sensor systemsdescribed herein can allow for at least three different electricalmeasurements to be performed: current, recovery voltage and open-circuitvoltage. Additionally, various embodiments of the disclosure relate toan (e.g., automated) system for performing the electrical measurements.Such systems may be further be configured to communicate the data toremote users. Aspects of exemplary sensor systems include:

-   -   Anode/cathode probe design capable of reconfiguration depending        on the characteristic of the site/environment (soils, sediments        and groundwater).    -   Cathode design allowing for exposure of the cathode to        atmospheric oxygen when the cathode is submerged in anaerobic        groundwater or saturated sediments/soils.    -   A monitoring system design that includes a device allowing for        one cathode (reference electrode) and one or more anodes        (measurement electrodes) for characterizing the chemical        (oxidizing/reducing) environment of the subsurface.    -   A monitoring system design that includes a device allowing for        an alternative reference electrode and one or more measurement        electrode for characterizing the chemical (oxidizing/reducing)        environment of the subsurface.    -   An anode located within a calibration chamber or module allowing        the capture of metabolic gases for the calibration of the        microbial sensor.    -   Measurement of open circuit voltage as a metric for the        determination of the chemical (oxidizing and reducing)        environment being monitored by the microbial sensing system.        (FIG. 6).    -   Measurement of the recovery voltage as a metric for the        determination of the substrate concentration, or substrate        turn-over rate. (FIGS. 7 and 8).

The sensor system may be employed in several different environments fora variety of scenarios including:

-   -   Remediation/Monitoring: The system can be used to characterize        contaminated (anaerobic) environments to determine the        efficiency of a remedial action.    -   Long-Term Monitoring: The system can be used for assessing        passive (Natural Zone Source Depletion) remediation projects, or        serve as a method for assessing Monitored Natural Attenuation        (MNA) where active remediation has been terminated.    -   Sentinel Monitoring: The system can be deployed in        uncontaminated (aerobic) environments (e.g., aquifers) to        determine if the aquifer was impacted by a release of        environmental contamination (i.e., petroleum fracking        operations, landfills). The presence of oxidizable environmental        contaminants will cause natural waters to change from aerobic to        anaerobic conditions creating a measurable voltage.

Sensor Measurements

The measurement of OCV provides a very different characterization of anenvironment surrounding the anode, or measurement electrode, than themeasurement of electrical current. The OCV mode of operation requiresthat no or an immeasurable amount of electrons (or electrical current)flow between the anode and cathode. (FIG. 6). The OCV measurement isused for the determination of the reduction/oxidation conditions, andnot the substrate concentration of the solution. The difference betweenthe OCV and current measurements is best explained by the response ofthe microbes to either: 1) a flow of electrons generated by theoxidation (source of electrons) of substrates at the anode and the flowof electrons to the cathode (the electron acceptor), or 2) the scenariowhere no electrons are allowed to flow from the anode to the cathode.

Certain types of microbes (e.g., Geobacter sp.) are attracted to andattach to the anode. The microbes form a biofilm that connects the cellsto the anode. The microbes can use several types of mechanisms to passthe electrons from the microbe to the anode through the biofilm. Theelectron transfer methods include nanowires, cytochromes and mobileshuttles. If electrons are allowed to flow from the anode to thecathode, the electrons produced during the oxidation of the substratesuse the electron transfer mechanisms and the flow of current is a metricfor the substrates oxidized by the microbes. The magnitude of theelectrical current may be in the lower microampere (μA) to nanoampere(nA) range for solutions with low substrate concentrations.

However, if the flow of current is terminated between the anode andcathode, the microbes begin to store the electrons generated by theoxidation of the substrate in temporary electron acceptors such ascytochromes. The cytochromes are located internal or external of themicrobes. The cytochromes located external of the microbes are embeddedin the biofilm. The OCV increases between the anode and the cathode asthe charge stored in the cytochromes increases. Voltages (OCV) of up to0.9 Volts are observed between an anode located in (e.g., completely)anaerobic conditions and a cathode exposed to atmospheric oxygen. Themicrobes continue the metabolism of the substrates and transfer thecharge into the temporary electron acceptors until the transfer ofcharge is no longer thermodynamically favorable. The microbes willmaintain the voltage between the anode and cathode until either 1) theflow of current is reestablished between the anode and cathodedischarging the stored charge, or 2) an alternative electron acceptor ispresent in the solution discharging the charge stored in the temporaryelectron acceptors (cytochromes).

Generally, the most significant electron acceptor that may be present inan environment is dissolved oxygen. If the concentration of dissolvedoxygen increases in the solution surrounding the anode, the storedcharge is transferred from the cytochromes to the dissolved oxygen. Thetransfer of charge from the temporary electron acceptors in the microbesand surrounding biofilm to the dissolved oxygen results in a decrease ofthe OCV measured between the anode and cathode. (FIG. 6). The OCV is lowin aerobic conditions and significantly higher is anaerobic conditions.If dissolved oxygen is removed from the solution and anaerobicconditions reestablished, the charge in the cytochromes increases with acorresponding increase in the OCV. Therefore, the OCV is a metric forthe chemical (reduction/oxidation) environment of the solution. This isa quite different than the estimate of substrate concentration that ismeasured by the flow of current between the anode and cathode.

Two primary problems are encountered with the measurement of constantcurrent between the anode and cathode as a metric for determiningsubstrate concentrations, or turn-over rates (at very lowconcentrations). The two problems are: 1) the increase in the internalresistance of the electrochemical cell over time, and 2) the complexityof the measurement circuitry required for the measurement of very lowelectrical currents. An increase in the internal resistance between theanode and cathode is observed over time when measuring constantcurrents. This results in a long-term drift of the signal toward lowercurrents. An analytical system that varies over time is difficult touse, unless a method of calibration can be devised to account for thechanging signal. Because most of the measurements envisioned to beperformed by the analytical system are at the level of the turn-overrates, the introduction of calibration standards is not appropriate. Onemethod of calibration, in accordance with the present disclosure, is themeasurement of metabolic gases (CO₂) produced at the anode by microbes(e.g., Geobacter, sp.).

In accordance with various embodiments of the disclosure, a method ofdetermining substrate concentration, or turn-over rates, uses recoveryvoltages as opposed to the measurement of constant current. The use ofrecovery voltage has two primary advantages including 1) changes ininternal resistance appear to be less of a factor, and 2) themeasurement of voltages—e.g., in the range of 0.3 to 0.6 Volts uses lesscomplex circuitry than used in the measurement of the low currents (uAto nA). However, the measurement of recovery voltage is a kineticmeasurement and the data analysis of recovery voltage is more complexthan the measurement of constant current.

The measurement of recovery voltage includes a three-step process. Thefirst step allows the OCV to stabilize. The second step uses a temporarydischarge of the charge stored in the cytochromes. The third step usesthe termination of the temporary discharge of charge, and a record ofthe increase of voltage over time as the cytochromes recover the chargelost in the second step.

During the first step, the OCV stabilizes at a constant or presetvoltage. In anaerobic conditions, the voltage will typically stabilizebetween 0.5 to 0.8 Volts.

After the OCV stabilizes, the second step allows the flow of electricalcurrent between the anode and cathode for a period of time. The flow ofcurrent partially discharges the charge stored by the cytochromes. Oneto ten minutes is a typical period of time to flow the current betweenthe anode and cathode. The voltage between the anode and cathode dropssignificantly (e.g., 0.2 to 0.5 Volts) when the current flows betweenthe anode and cathode.

After the period of time, the third step terminates the flow of currentbetween the anode and cathode. After the flow of current is terminated,the OCV increases. The OCV voltage versus time curve generated issimilar in characteristics to the voltage-time curve generated duringthe charging of a capacitor, as illustrated in FIG. 7. The charge storedunder the voltage-time curve is directly related to the electronsgenerated from the oxidation of substrates. Therefore, the generationand measurement of the recovery voltage may be used to determine eithersubstrate concentration or turn-over rate (at lower concentrations).

Sensor Design

The microbial fuel cell designs for the production of energy aretypically optimized for the production of electrical current. Theoptimization of electrical current includes increasing surface areas ofthe anode and cathode, flow-through reactors to increase the substrateconcentrations exposed to the anode and other factors to increase theproduction of electrical current

In contrast to the measurement of BOD at treatment facilities where thewater is transported in pipes, most environmental monitoring isperformed by the insertion of probes or sensors directly into anenvironment, such as soils or into groundwater monitoring wells.Therefore, for these applications, microbial sensors are desirablyconfigured as probes and have the flexibility to be deployed in avariety of natural environments including soils, sediments and naturalwaters.

In accordance with various embodiments of the disclosure, a cathode canbe submerged in anaerobic (e.g., soil and groundwater) environments.Systems for introducing atmospheric oxygen to the cathode, when thecathode is exposed to submerged anaerobic environments, include aconduit or passage through the interior of the probe allowing theintroduction of atmospheric oxygen to the cathode. In these cases, aninterior of a probe can be hollow and a cathode assembly can befabricated into a port located in the wall of the probe. The cathodeport is located below the static water level. Atmospheric oxygen passesfrom the atmosphere through a conduit tube (or “snorkel”) into thehollow interior of the cathode probe. The cathode assembly is typicallycomposed of a polymer membrane and a cathode. The cathode is typically aplatinum impregnated carbon cloth. The polymer membrane of the cathodeassembly can perform two tasks: 1) prevent water from entering theinterior of the probe, and 2) allow oxygen present in the interior ofthe cathode probe to diffuse through the polymer membrane to the cathodeexposed to anaerobic environment, i.e., groundwater. Additionally oralternatively, a waterproof film or layer may be applied to the cathodecloth.

Because of the myriad of potential environmental applications to monitorsoils and ground waters, the positional relationship of the anode andcathode may be changed based on a particular application. Theflexibility of the various probes and systems described herein allowsfor connection of the anode and cathode (or reference and measurementelectrodes) to alternative deployment assemblies including floats formonitoring wells. The anode/cathode/electrode connectors include pipethreads, other quick disconnect connectors, and the like.

Two situations illustrating the desire for flexibility in assemblinganode and cathode (or, generally electrodes) for deployment in the fieldinclude 1) direct insertion probes for monitoring soils, and 2)deployment of the anode and cathode (electrodes) in monitoring wells.The first application allows an anode to be directly connected to acathode. The assembled probe may be directly inserted into the submergedsoil/sediments. The second application allows for the monitoring ofgroundwater. The cathode is connected to floating module located at thestatic water level within the interior of the well while the anode islocated at a submerged location below the floating module within thewell. The cathode assembly connects to the floating module and thisconnection insures the cathode is both submerged below the static waterlevel, and that the cathode has a direct conduit or snorkel toatmospheric oxygen.

The measurement of OCV, as opposed to electric current, allows for asensor design using one cathode as a reference electrode and one or moreanodes as measurement electrodes. The deployment of multiple sensorarrays allows for the characterization the chemical (aerobic/anaerobic)conditions of a site. (FIG. 6). The measurement is made possible becauseno current flows between the electrodes in the system.

The OCV mode of operation in accordance with various embodiments of thedisclosure allows for use of alternative reference electrodes in lieu ofa cathode. The cathode is defined as a reference electrode exposed to anoxidizing environment, such as oxygen present in air as the ultimateelectron acceptor. For example, if three anodes in an environment aremeasured against a common cathode in the OCV mode of operation, threedifferent voltages are measured (Table 1). In the example below, anode#3 is the located in the deepest location of test chamber and thereforeis located in the most anaerobic conditions.

TABLE 1 Anode #1 Anode #2 Anode #3 .397 volts .321 volts .713 volts

If it is determined that voltage of Anode #3 is not likely to changeduring the timeframe of an investigation of a site, Anode #3 may beselected as an alternative reference electrode because it is located ina stable anaerobic location. If Anode #3 is selected as the referenceelectrode, the OCV of two remaining anodes are measured against Anode #3(Table 2).

TABLE 2 Anode #1 Anode #2 Anode #3 .316 .392 NA

Therefore, the cathode (electrode exposed to oxygen, aerobic conditions)is replaced as the reference electrode with Anode #3 exposed to stableanaerobic conditions, the alternative reference electrode. Thereplacement of the reference electrode results in a similarcharacterization of the site in determining the chemical (reducing oroxidizing conditions) environmental of the site. However, thereplacement of the cathode as the reference electrode results in thecharacterization of the site using a much simpler analytical system.Because the cathode is typically the most complex component of a sensorarray, its elimination increases the life expectancy of the sensorarray. The alternative electrode can be an inert electrode (inert withregard to the surrounding environment) composed of, for example,graphite or other conducting material not corroded in the environment.The design of the alternative reference electrode can be similar to orthe same as the anode design.

Referring to the drawing figures, FIG. 1 illustrates, in cross-sectionalview, an exemplary microbial sensing system 100 in accordance with atleast one embodiment of the disclosure. An anode assembly 6 includes aninert anode 12 connected to an anode body 14. The inert anode 12 can becomposed of, for example, graphite or other conducting material—e.g.,that does not corrode in the environment in which the anode is inserted.The anode body 14 is composed of a polymer or other non-conductingmaterial not corroded in the environment the anode body 14 is inserted.A temperature probe (or sensor) 16 is embedded within the inert anode12. The temperature probe 16 may be a thermocouple, thermistor, or RTD.An anode cable 18 electrically connects to the anode 12. A temperatureprobe cable 20 electrically connects to the temperature probe 16. Ananode connection port 22 is located at the terminal end of anode body 14allowing the connection with an anode connector 10. The anode connector10 is a hollow tube with a method of connection to the anode connectionport 22. The method of connection can include, for example, threads orother type of quick-disconnect fittings. The anode connector 10 allowsthe passage of the anode cable 18 and the temperature cable 20 from theanode assembly 6 to a cathode assembly 8. The anode connector 10 iscomposed of a polymer or other non-conducting material not corroded inthe environment the anode connector 10 is inserted.

The anode connector 10 connects to a lower cathode connection port 24located at the terminal end of a cathode body 26. The cathode body 26 iscomposed of a polymer or other non-conducting material not corroded inthe environment the cathode body 26 is inserted. An interior 30 of thecathode body 26 is a hollow tube allowing the passage of the anode cable18 and the temperature cable 20 through the cathode assembly 8.

A cathode mounting port 28 passes through the cathode body 26 andconnects the hollow interior 30 to an exterior environment 48. Thecathode mounting port 28 is fitted with a gas-permeable membrane 32. Thegas-permeable membrane 32 is composed of a polymer film or othermaterial selected to allow the diffusion of oxygen through the membraneand prevent the introduction of water into the hollow interior 30. AnO-ring 34 is fitted over the permeable membrane 32. A cathode 36 and acathode frame 38 are fitted over the O-ring 34. Mounting screws 40, 42secure the cathode frame 38 and the cathode 36 to the cathode body 26using the mounting screw holes 44, 46 in the cathode body 26. Themounting screws 40, 42 are used to apply pressure on the O-ring 34 tocreate a water-tight seal between the O-ring 34 and the gas-permeablemembrane 32 preventing water from entering the hollow interior 30 of thecathode body 26.

The cathode 36 is composed of a platinum-coated carbon fabric or othermaterial capable of reducing molecular oxygen with the electronsgenerated at the anode 12. The cathode frame 38 is composed of a polymeror other non-conducting material not corroded in the environment thecathode frame 38 is inserted.

A cathode wire 50 passes through the wall of the cathode body 26 andelectrically connects the cathode 36 with a conductive collar 52 locatedwithin the hollow interior 30 of the cathode body 26. A cathode cable 54connects to the conductive collar 52.

An upper cathode connection port 56 is located at the terminal end ofthe cathode body 26 connects with a snorkel (also referred to herein asconduit) 58. The snorkel 58 is a hollow tube with a method of connectionto the upper cathode connection port 56. The method of connectionincludes threads or other type of quick-disconnect fitting. The snorkel58 allows the passage of the anode cable 18, temperature cable 20 andthe cathode cable 54 from the cathode assembly 8 to the atmosphere 60.In addition, the snorkel 58 provides a conduit for oxygen from theatmosphere 60 to diffuse into the hollow interior 30 of the cathode body26. The oxygen in the hollow interior 30 diffuses through thegas-permeable membrane 32 to the cathode 36. The snorkel 58 is composedof a polymer or other non-conducting material not corroded in theaqueous environment 48 (the environment in which the measurementelectrodes are placed is also referred to herein as a first environment.The anode cable 18, temperature cable 20, and the cathode cable 54connect to a measurement module 59, which can include a device tomeasure OCV and/or RV, and optionally current as described herein. Thedevice can include one or more circuits.

In the operation of the illustrated system, selected species ofbacteria, such as Geobacter sp., form biofilms on the surface of theanode 12. The bacteria oxidize various substrates in the aqueousenvironment 48 and transfer the electrons in the oxidation reactionthrough the biofilms to the anode 12. The electrons flow from the anode12 through the anode cable 18 to a measurement module 59 located atground surface 82. The electrons pass from the measurement module 59(and the device) through the cathode cable 20 and the conductive collar52 to the cathode wire 50. The electrons flow through the cathode wire50 into the cathode 36 located in the aqueous environment 48. Theelectrons reduce the dissolved molecular oxygen in the aqueousenvironment 48 to water at the cathode 36. Molecular oxygen serves asthe ultimate electron acceptor for the microbial sensor system. Ifmolecular oxygen is not present in the aqueous environment 48 in thevicinity of the cathode 36, an alternative path is provided allowingatmospheric oxygen to be present in the vicinity of the cathode 36.Molecular oxygen diffuses from the atmosphere 60 through the snorkel 58into the hollow interior 30 of the cathode body 26. The molecular oxygenin the hollow interior 30 of the cathode body 26 diffuses through thegas-permeable membrane 32 to the cathode 36.

In exemplary system 100, the anode assembly 6 and cathode assembly 8 canbe quickly reconfigured using the anode connector 3 and the snorkel 58.The anode body 14 has the anode connection port 22 with either threadsor other type a quick disconnection. The cathode body 26 has the lowercathode connection port 24 with the threads or other means of quickdisconnection. The anode connector 3 has threads or other type of quickdisconnection and provides the connection between the lower cathodeconnection port 24 and the anode connection port 22. The anode connector10 is hollow to allow the passage of the anode cable 18 and thetemperature cable 20 into the hollow interior 30 of the cathode body 26.The design allows different lengths of anode connectors 10 to beinserted or attached. Additionally or alternatively, the anode assembly6 may be quickly exchanged with different configurations of the anodeassembly 6.

The upper terminal end of the cathode body 8 includes the upper cathodeconnection port 56 with threads or other type of quick disconnectfeatures. The snorkel 58 has a terminal end designed to connect with thethreads or other quick disconnect features of the upper cathodeconnection port 56. The interior of the snorkel 58 is hollow allowingthe passage of the anode cable 18, cathode cable 54 and temperaturecable 20 from the hollow interior 30 of the cathode body 26 through thesnorkel to a measurement module 59 at the surface 82. The design allowsthe quick and efficient method of reconfiguring the anode assembly 6,cathode assembly 8, and the snorkel 58 into many variants to match thedesired configuration for site conditions.

FIG. 2 illustrates another exemplary microbial sensor system 200 with analternative design of the anode assembly. In this embodiment, the anodeis located within a calibration chamber or module 62. The system can beformed by reconfiguring a cathode assembly with an alternative design ofthe anode assembly. Microbial sensor system 200 captures metabolicgases, such as carbon dioxide, generated within a calibration chamber62, such that microbial sensor system 200 can be calibrated. Thecalibration chamber 62 is composed of a polymer or other non-conductingmaterial not corroded in the aqueous environment 48. A gas tube 68 isused to remove the metabolic gases generated within the calibrationchamber 62. An inert anode 12 is located within the calibration chamber62. A one-way check valve 66 connects to an entrance port 64 of thecalibration chamber 62. The one-way check valve 66 is composed of apolymer or other material not impacted by the (e.g., aqueous)environment 48. The one-way check valve 66 is orientated to allow theflow of water into the calibration chamber 62. The anode connector 10connects the calibration chamber 62 with the cathode assembly 8. Thesnorkel or conduit 58 connects to the upper port of the cathode assembly8. The terminal end of the snorkel 58 is exposed to the atmosphere 60(e.g., air), also referred to herein as a second environment. The anodecable 18 electrically connects to the anode 12. The anode cable 18passes through upper wall of the calibration chamber 62, the anodeconnector 10, the cathode assembly 8, and the snorkel 58 to themeasurement module 59. The gas tube 68 passes through the upper wall ofthe calibration chamber 62, the anode connector 10, the cathode assembly8, and the snorkel 58 to the atmosphere 60.

The operation of the system 200 is performed by measuring the differencein the carbon dioxide generated within the calibration chamber 62 whencurrent is allowed to flow between the anode 12 and cathode 36 versuswhen no current is allowed to flow between the anode 12 and cathode 36.The moles of carbon dioxide generated are compared with the number ofmoles of electrons passing from the anode 12 to the cathode 36.

The carbon dioxide generated accumulates in the calibration chamber 62.The gas tube 68 is used to remove the carbon dioxide gas from thecalibration chamber 62 for measurement of the volume of the gasgenerated within the calibration chamber 62. The withdrawal of thecarbon dioxide can be performed using a syringe or other method ofcollection the gas sample from the calibration chamber 62. Theevacuation of the gas from the calibration chamber 62 opens the one-waycheck valve 66 allowing water 48 to flow into the calibration chamber62.

FIG. 3 illustrates another microbial sensing system 300, suitable fordeployment in, for example, a monitoring well. Microbial sensing system300 can be formed by, for example, reconfiguration of the anode andcathode assemblies of microbial sensing system 100. The cathode assembly8 connects to a float 76 using the snorkel 58. The float 76 is disposedwithin a well casing 80 to be monitored. The float 76 is located at thestatic water level 78 within the well casing 80. The float assembly 76allows oxygen in the atmosphere 60 to diffuse through the snorkel 58 andinto the cathode assembly 8. The float 76 is composed of a polymer orother non-conducting material not corroded in the aqueous environment48. The cathode cable 54 electrically connects to the cathode assembly 8and passes through the snorkel 58 to a control/communication module 92located at the surface 82.

In the illustrated example, the anode assembly 6 is located in theaqueous environment 48 below the static water level 78 within the wellcasing 80. The anode cable 18 electrically connects to the anodeassembly 6. The anode cable 18 passes through the float 76 to thecontrol/communication module 92 located at the surface 82.

The operation of the microbial sensing system 300 deployed in amonitoring well can insure that atmospheric oxygen is available to thecathode regardless of the water level with the monitoring well. Theanode assembly 6 and cathode assembly 8 are connected to the bottomsurface of a float 76. The position of the float 76 within the wellcasing 80 can depend on the static water level 78 of the aquifer. Thevertical movement of the float 76 within the monitoring well facilitatesthe upper port of the snorkel 58 exposure to the atmosphere 60. Theoxygen in the atmosphere 60 diffuses through the snorkel 58 to cathodeassembly 8.

The anode assembly 6 suspended below the float 76 can determine achemical nature or a substrate concentration of the aqueous environment48. Alternatively, the anode assembly may be deployed within the wellcasing 80 below the static water level 78 without suspending the anodeassembly 6 from the float 76. The electrons, produced by the microbes onthe surface by oxidation reaction, from the anode 6, flow through theanode cable 18 to the control/communication module 92. The electronsflow from the control/communication module 92 through the cathode cable54 to the cathode assembly 8. The flow of electrons is related to thesubstrate concentration, or turn-over rate, in the vicinity of the anode6. Alternatively, the current flow between the anode assembly 6 andcathode assembly 8 is terminated and the measurement of the open-circuitvoltage is a metric for the chemical environment (oxidizing or reducing)in the vicinity of the anode assembly 6.

FIG. 4 illustrates a multiple microbial sensor network or array 400 withthe cathode assembly 8 used as the reference electrode. Several anodes(or measurement electrodes) 6, 84, 86 are located below a static waterlevel 78. The cathode assembly 8 is connected to the snorkel 58. Theupper terminal end of the snorkel 58 is exposed directly to theatmosphere 60, or the soil-gas located between the static water level 78and the ground surface 82. The cathode cable 54 electrically connectsthe cathode assembly 8 with a control/communication module 92 located atthe surface 82. An anode cable 18 electrically connects the anode 18with the control/communication module 92 located that the ground surface82.

The operation of network 400 allows a field deployment of multipleanodes using one cathode as the reference electrode to determine thechemical (oxidizing or reduction) environment of a site or anenvironment. The cathode assembly 8 is located below the static waterlevel 78 in the aqueous environment 48. The snorkel 58 is connected tothe cathode assembly 8, allowing the diffusion of oxygen in theatmosphere to reach the cathode assembly 8. A cathode cable 54 connectsthe cathode assembly with the control/communication module 92 located atground surface 82. Multiple anodes 6, 84, 86 are placed at differentlocations within the aqueous environment 48 to be characterized. Theanodes 6, 84, 86 are connected with anode cables 18, 88, 90 to thecontrol/communication module 92 located at, for example, ground surface.The open-circuit voltage between the cathode assembly 8 and to each ofthe anodes 6, 84, 86 is measured to determine the chemical (oxidizing orreducing) nature of the aqueous environment 48 in the vicinity of theanodes 6, 84, 86.

Referring to FIG. 5, a multiple microbial sensor network or array 500,with an inert electrode 94 located in a stable anaerobic location of theaqueous environment 48, is illustrated. If the aerobic conditions arestable in the vicinity of the inert electrode 94, the electrode is useda reference electrode. Multiple anodes 6, 84 are located in the aqueousenvironment 48 below a static water level 78. An electrode cable 96electrically connects the inert electrode 94 with thecontrol/communication module 92 located at the surface 82. An anodecable 18 electrically connects the anode 6 with thecontrol/communication module 92 located that the ground surface 82.

The operation multiple microbial sensor network 500 can be employed in afield deployment of multiple measuring electrodes 6, 84 and using aninert reference electrode 94 in determining the chemical (oxidizing orreduction) environment of the site. The reference electrode 94 is placedin an anaerobic location on the site that is not expected to change (ornot significantly change) during the course of an investigation. Anelectrical cable 96 connects the reference electrode with thecontrol/communication module 92 located at, e.g., ground surface 82.Multiple anodes 6, 84 are placed at different locations within theaqueous environment 48 to be characterized. The anodes 6, 84 areconnected with anode cables 18, 88 to the control/communication module92 located at ground surface. The open-circuit voltage between thereference electrode 96 and to each of the anodes 6, 84 is measured todetermine the chemical (oxidizing or reducing) nature of the aqueousenvironment 48 in the vicinity of the anodes 6, 84.

FIG. 6 illustrates OCV versus time for three electrodes (e.g., anodes)disposed within a test chamber, wherein each electrode is at a differentlevel within the test chamber. Oxygen was periodically introduced intothe reaction chamber. As the oxygen is introduced, the OCR is reduced,indicating an aerobic environment in the test chamber. Conversely, whennitrogen is introduced into the test chamber, the OCR increases,indicating an anaerobic environment.

FIGS. 7 and 8 illustrate RV measurements in Volts taken from threemeasurement electrodes versus time in minutes. The RV can be obtainedusing the techniques described above. FIG. 8 is a log-log plot of thedata illustrated in FIG. 7. The area under the curve in FIG. 8corresponds to a concentration of a substrate in the environment.

The exemplary systems, probes, and methods have been described abovewith reference to a number of exemplary embodiments and examples. Itshould be appreciated that the particular embodiments shown anddescribed herein are illustrative of the invention and its best mode andare not intended to limit in any way the scope of the invention. It willbe recognized that changes and modifications may be made to theexemplary embodiments without departing from the scope of the presentinvention. These and other changes or modifications are intended to beincluded within the scope of the present invention. Further, the subjectmatter of the present disclosure includes all novel and nonobviouscombinations and subcombinations of the various processes, systems,arrays, and probes, and other features, functions, acts, and/orproperties disclosed herein, as well as any and all equivalents thereof.

We claim:
 1. A method of measuring microbial activity in an environment,the method comprising the steps of: providing a reference electrode inthe environment; providing one or more measurement electrodes in theenvironment; and measuring one or more of an open circuit voltagebetween the reference electrode and the one or more measurementelectrodes and a recovery voltage between the reference electrode andthe one or more measurement electrodes to characterize the environment.2. The method of measuring microbial activity in an environment of claim1, wherein the step of measuring comprises measuring one or more of theopen circuit voltage between the reference electrode and each of aplurality of measurement electrodes and the recovery voltage between thereference electrode and each of the plurality of the measurementelectrodes.
 3. The method of measuring microbial activity in anenvironment of claim 1, wherein the reference electrode comprises aninert electrode.
 4. The method of measuring microbial activity in anenvironment of claim 1, further comprising a step of measuring a currentbetween the reference electrode and each of the one or more measurementelectrodes.
 5. The method of measuring microbial activity in anenvironment of claim 1, further comprising providing the referenceelectrode access to another environment using a conduit.